CAD is the major public disorder in both developed and developing countries(Amrein, et al., 2020; Benjamin, 2018), among them, atherosclerosis(AS) as the main pathological mechanism occupies an important number. It is well-known that VSMCs are one of the main cell types involved in the development of atherosclerosis. In response to detrimental stimuli, VSMCs undergo phenotypic conversion, including abnormal proliferation and migration, giving rise to intimal hyperplasia and accelerating the progression of atherosclerosis(Owens, et al., 2004). Therefore, revealing the molecular mechanism of VSMCs involved in atherosclerosis may improve the unsatisfactory diagnosis and treatment of CAD.
M6A RNA methylation is the largest proportion of RNA modification in eukaryotes(Zhou, et al., 2017). Apart from the regulation of mRNA, m6A methylation likewise controls the biological functions of ncRNAs, especially many lncRNAs(Jakobi, et al., 2020; Patil, et al., 2016; Yang, et al., 2018; Yang, et al., 2017). M6A modifications in VSMCs have been demonstrated in previous studies, which played a critical role in atherosclerotic lesion restenosis(Zhu, et al., 2020). As far as we know, our study investigated the m6A-lncRNA landscape in HCASMCs by using RNA MeRIP-Seq. The findings revealed the dynamic characteristics of m6A modification in HCASMCs, which is closely related to the pathogenesis of CAD.
We carried out genome-wide maps of m6A-flagged lncRNAs between the two groups by MeRIP-seq. We compared the expression of m6A modified lncRNAs and non-m6A modified lncRNAs between the two groups. We found that m6A-level in lncRNAs has a positive relation with the expression level of lncRNAs. This may be that m6A changes the stability of lncRNAs, reduces the degradation of lncRNAs, and thus increases its expression. Moreover, m6A modification of lncRNAs can affect the expression and regulation of downstream molecules by changing their secondary structure, molecular stability, splicing, or degradation(Chen, et al., 2021).
GO and KEGG analyses of the associated genes of lncRNAs with different m6A levels were subsequently performed to explore their potential function of the associated genes. It is also the first time to clarify the role of differential m6A levels in lncRNAs with a further influence on the biological behaviors of HCASMCs. GO and KEGG analyses revealed that regulation of small GTPases mediated signal transduction, adherens junction, fatty acid metabolism, rap1and ErbB signaling pathway may be participating in atherosclerosis.It is well-known that readjustment of the actin cytoskeleton is essential for cell shape changes and is regulated by multiple cellular proteins. There into, small GTPases are characterized as modulators of the actin cytoskeleton and take a vital role in cell migration. Small GTPases, divided into five families(Wennerberg, et al., 2005), among them, the Rho GTPase family is a core regulator of actin reorganization that is closely related to cell migration(Ridley and Hall, 1992). Over the years, Rho is a known therapeutic target for CAD. In addition, Rap1, as a regulator of cell proliferation, it is obvious that this small GTPase takes a very crucial role in the cardiovascular disorder-related signaling pathways(Jeyaraj, et al., 2011). In addition, recognized in a variety of cells and tissues, the ErbB signaling pathway can control cell proliferation and migration by mediating the MAPK, JAK/STAT pathway, and PI3K/Akt pathway(Kuroshima, et al., 2020). These analysis results suggest that the differential m6A site of lncRNAs may be an important breakthrough point to control the phenotypic switching of VSMCs in atherosclerosis, which will give a distinctive approach for the treatment of CAD. Our findings provided a resource of differentially expressed profiles of lncRNA m6A for revealing the mechanism of m6A modification in CAD.
The above prediction of biological function revealed that the associated genes of lncRNAs with different m6A levels are closely connected to the pathways related to phenotypic switching of VSMCs. For ascertain the reliability of sequencing data, eighteen lncRNAs were screened out for qRT-PCR verification, based on foldchange and P-values. We found that seven lncRNAs were stably downregulated in PM group compared with control group, that is consistent with the sequencing data. Moreover, we found differentially downregulated methylation peaks in the two lncRNAs from sequencing data. A hint could be obtained that these lncRNAs with different levels of m6A modification may be more distributed and function in the cytoplasm. Normally, Generally, lncRNAs located in the cytoplasm can participate in normal physiological activities and abnormal pathological activities by changing the stability of mRNA, regulating translation events(Zhao, et al., 2018), sequestering miRNAs(Zhang, et al., 2017), and acting as scaffolders for protein complexes(Kotake, et al., 2011), stability of ribonucleoprotein complexes(Zhao, et al., 2016), protein phosphorylation, and activation of signaling pathways(Chen, et al., 2020; Noh, et al., 2018). Undoubtedly, there is still a long way to go to further clarify the regulatory mechanisms and biological functions of m6A modification on lncRNAs in CAD. We will conduct more in-depth research in the future to address these issues and we are optimistic that the continued study of the noncoding transcriptome holds outstanding promise to impact our understanding of normal physiology and diseases. Undoubtedly, more in-depth studies are needed to clarify the role of m6A modification on lncRNAs during the occurrence and development of CAD, and the continuous study of noncoding transcriptomes will also shed light on the differences between normal physiology and disease states from different perspectives, to improve the diagnosis and treatment of CAD.